Peroxisomal Disorders are a newly recognized group of inherited neurological diseases caused by dysfunction of peroxisomes. Pathognomonic amounts of phytanic acid, a highly branched fatty acid, accumulates in a number of peroxisomal diseases. We have found that phytanic acid is alpha-oxidized to pristanic acid in peroxisomes in humans, and that this activity is deficient in peroxisomes isolated from cultured skin fibroblasts from patients with Refsum Disease and Rhizomelic Chondrodysplasia Punctata. However, the molecular mechanisms of the various enzymatic steps in alpha-oxidation of phytanic acid and the molecular basis of the abnormality in phytanic acid metabolism in peroxisomal diseases remain to be elucidated. The proposed studies will focus on the understanding of the mechanisms of formation of alpha-hydroxyphytanic acid, alpha-ketophytanic acid, intermediates in the alpha-oxidation of phytanic acid, to pristanic acid in Specific Aim 1. Our discovery of the presence of a complete alpha- oxidation system for phytanic acid in peroxisomes provides an opportunity to carry out these studies in purified peroxisomes. Specific Aim 2 is proposed to clone the cDNA for phytanic acid alpha-hydroxylase, an enzyme with deficient activity in Refsum Disease, and to determine its chromosomal localization. Specific Aim 3 is devoted to the investigation of the molecular basis of the abnormality in phytanic acid metabolism in Refsum Disease. This will be facilitated by quantitation of the levels of transcriptional (m-RNA) and translational (proteins) products of the phytanic acid alpha-hydroxylase gene in Refsum Disease tissues and the identification of a specific abnormality in the cDNA sequence for phytanic acid alpha-hydroxylase from Refsum Disease as compared to control. The possible normalization of the activity for phytanic acid alpha-hydroxylase/alpha-oxidase activities in cells from Refsum Disease will be investigated following transfection of cDNA for phytanic acid alpha-hydroxylase. Because of the short history of peroxisomes, relatively little is known about their metabolic biology and associated pathophysiology as compared to other organelles. The proposed studies are significant and will utilize state of the art methodologies in Cellular and Molecular Biology to enhance our understanding of this newly discovered enzyme system in peroxisomes. The molecular basis of the pathogenesis in Refsum Disease will be investigated as a prototype for diseases with phytanic acid metabolic abnormalities.